Br. J. Pharmacol. (1990), 99, 503-508

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Macmillan Press Ltd, 1990

Opioid receptor ligands in the neonatal rat spinal cord: binding and in vitro depression of the nociceptive responses I.F. James, J. Bettaney, M.N. Perkins, S.B. Ketchum & A. Dray Sandoz Institute for Medical Research, 5 Gower Place, London WC1E 6BN 1 Opioid receptors in the neonatal rat spinal cord have been characterized by measurements of ligand binding to crude membrane fractions and by functional tests on the nociceptive spinal response in a spinal cord-tail preparation in vitro. 2 There were high affinity binding sites for [3H]-[D-Ala2, MePhe', Gly(ol)5]enkephalin (DAGOL), [3H]-U69593, and [3H]-ethylketocyclazocine (EKC) on spinal cord membranes from neonatal rats. Hill slopes for binding of [3H]-DAGOL and [3H]-U69593 were close to unity. The Hill slope for binding of [3H]-EKC was less than unity, even after its interactions at p-receptors had been blocked with 100nm unlabelled DAGOL. Binding sites for [3H]-[D-Pen2, D-Pen5]enkephalin (DPDPE) could not be detected. 3 In competition assays U50488 was as potent as PD1 17302 and U69593 in competition for either [3H]U69593 or [3H]-EKC binding sites. Hill slopes for a range of competing ligands at [3H]-DAGOL or [3H]-U69593 sites were close to unity. Hill slopes for competition at [3H]-EKC sites were less than one. 4 In the spinal cord-tail preparation from neonatal rats, opioid receptor agonists depressed spinal nociceptive responses evoked by application of capsaicin or heat to the tail. The order of potency was DAGOL > U69593 = PD117302 > morphine > U50488 = [D-Pen2, L-Pen5]enkephalin (DPLPE). 5 The antagonist naloxone was about equally potent against DAGOL, morphine and DPLPE, and about ten times less potent against U69593 and PD1 17302. The effects of U50488 were much less sensitive to blockade by naloxone than the effects of PD1 1703 or U69593. The antagonist, nor-binaltorphimine was equipotent against all three K agonists. 6 The absence of 6-binding sites, and the low potency and relatively high sensitivity to naloxone suggest that DPLPE could be working at p-receptors in the neonatal rat spinal cord. 7 The binding assays show that U50488 has the same affinity as PD1 17302 and U69593 for K-receptors, yet it was less effective in the depression of nociceptive responses. This may be because U50488 has a relatively low efficacy at K-receptors. It is possible that at high concentrations U50488 activates receptors not affected by other K-ligands. These additional receptors may be non-opioid receptors (hence the insensitivity to naloxone), or they could be a subtype of K-opioid receptor. K

Introduction There are at least three types of opioid receptor, p, 6 and K, in the adult mammalian brain (Moskowitz & Goodman, 1984; Mansour et al., 1987) and spinal cord (Zarr et al., 1986; Traynor & Wood, 1987). Functional studies made in adult spinal cord support the involvement of y- (Schmauss & Yaksh, 1984) and 6- (Porecca et al., 1984; Rodriguez et al., 1986) opioid receptors in spinal antinociception. Evidence for the involvement of K-receptors is less clear, as K-ligands are inactive in some tests (Schmauss & Yaksh, 1984; Leighton et al., 1988), but active in others (Schmauss & Yaksh, 1984; Porreca et al., 1987). The study of drug effects on the spinal cord in vivo can be complicated by penetration into the brain or systemic redistribution of drugs given by the intrathecal route (McQuay et al., 1989). One way round this is by use of functionally relevant models maintained in vitro. In the neonatal rat, cutaneous nociceptors are well developed and have functional characteristics similar to those found in adult animals (Fitzgerald, 1985; 1987). These nociceptors respond to a variety of noxious peripheral stimuli (Yanagisawa & Otsuka, 1984; Fitzgerald, 1985; 1987) and nocifensor responses have been measured in vivo with behavioural tests (see McDowell & Kitchen, 1987 for references). A preparation from the neonatal rat has been developed in which the spinal cord with the tail attached can be maintained for prolonged periods in vitro (Yanagisawa & Otsuka, 1984; Yanagisawa et al., 1984). The activation of polymodal nociceptors in the tail can be measured by recording the depolarization produced in a spinal ventral root. Nociceptive mechanisms therefore are amenable to analysis in vitro. Nociceptive responses were depressed in this preparation by

administration of opioids to the spinal cord (Yanagisawa & Otsuka, 1984; Yanagisawa et al., 1984; Dray & Perkins, 1986), though the specific opioid receptors involved have not been studied. p- and K-receptors have been found in binding studies with neonatal rat brain (Spain et al., 1985; McDowell & Kitchen 1987), but sites on neonatal spinal cord were not measured. It seemed important, therefore, to determine the types of opioid receptors present in the neonatal spinal cord, and to evaluate their function in the antinociceptive effects of opioids. Some preliminary findings from this study have already been presented (Dray et al., 1989).

Methods

Ligand binding Spinal cords were removed from newborn or one day old rats and homogenized in 10 volumes of 50mM Tris-HCl, pH 7.4. A crude membrane fraction was isolated by centrifuging the homogenate at 1700Og for 20min. Membranes were resuspended in Hanks balanced salt solution, buffered to pH 7.4 with HEPES and left on ice for 30min to allow dissociation of any endogenous opioids. They were then centrifuged as above and washed twice in Tris buffer. Finally the membrane preparation was resuspended in Tris to give approximately 0.3 mg protein ml- '. Use of Tris buffer without additional ions is standard in measurement of opioid binding, because binding of agonists can be reduced quite significantly in the presence of physiological ions (see for example Childers et al., 1979; James & Goldstein, 1984). Tris was used in this

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study, firstly, to avoid potential reductions in binding and, secondly, to allow direct comparisons with other binding studies. The binding assay mixture contained 250y1 of membrane suspension plus appropriate radioligands (and unlabelled ligands where necessary) in a total volume of 500 P1 of Tris buffer. Samples were incubated at room temperature for 60min, filtered through Whatman GF/C glass fibre filters, washed three times with 5ml of ice-cold Tris buffer and counted. Saturable binding was the difference between binding in the absence and presence of 10.uM naloxone. [3H]-[D-Ala2, MePhe4, Gly(ol)5]enkephalin ([3H]-DAGOL), [3H]-[D-Pen2, D-Pen5]enkephalin (DPDPE) and [3H]-U69593 were used as selective p-, 3- and K-ligands respectively. [3H]-ethylketocyclazocine (EKC) was also used to label K-receptors after its binding to y sites was blocked with 100 nm unlabelled DAGOL. DPDPE was used as the 6-ligand in the binding assays because radiolabelled [D-Pen2, L-Pen5]enkephalin (DPLPE, which had been used in the functional assays) is not available. DPLPE and DPDPE are very similar in affinity and selectivity for p-, 3- and K-receptors (James & Goldstein, 1984). In competition assays, radiolabelled ligands were used at a concentration of 1 nM.

Functional studies The spinal cord and attached tail were removed from one day old rats following decapitation. The most superficial layer of the skin was removed from the distal four fifths of the tail with fine forceps. This procedure exposed cutaneous fibres and their endings, and allowed penetration of drugs. Damage to deeper skin layers and tissue severely compromised chemical sensitivity. The spinal cord and tail were superfused separately with a physiological salt solution (composition mM: NaCl 138.6, KCl 3.35, CaCl2 1.26, MgCl2 1.16, NaHCO3 21.0, NaHPO4 0.58, glucose 10) at 240C and gassed with 95% 02/5% CO2. The activation of peripheral fibres in the tail by peripheral stimuli was assessed by measuring the depolarization in a spinal ventral root (L3-L5). This was recorded by a low impedance extracellular glass micropipette, filled with electrolyte, placed in an electrolyte-filled well which contained the selected ventral root. The ventral root potential was recorded d.c. with respect to the spinal cord which was earthed. Signals were amplified by conventional methods and displayed simultaneously on an oscilloscope and on a rectilinear chart recorder. Noxious stimuli consisted of a brief (10s) exposure of the tail to a submaximal concentration of capsaicin (0.4-0.6upM) or a 10s exposure to superfusate heated to 480C. Innocuous stimulation consisted of lightly brushing the tail (10s) with a fine sable-hair paint-brush. Peripheral stimuli were repeated sequentially at intervals of 15 min. In preliminary studies, the spinal cord was treated with opioids for up to 60min, then the peripheral stimuli were tested. The effect of opioids stabilized after 15min exposure. This presumably reflects the time taken for the opioid to equilibrate in the tissue. Hence, in routine tests of the effects of opioid agonists, each ligand was administered in the spinal cord superfusate for at least 15 min before the effect of peripheral stimulation was tested. The concentration of each opioid was then increased to obtain a cumulative dose-effect relationship against capsaicin and heat-evoked responses. The agonist EC50 was obtained from a plot of percentage inhibition of the ventral root response against opioid dose. At least four concentrations of each opioid were tested in these experiments with one concentration producing a maximal effect. In the studies with opioid antagonists a similar protocol was used. Thus cumulative concentrations (at least three) of antagonist were administered in the presence of a fixed concentration of the agonist (usually twice the EC50 concentration). Antagonist potency was assessed by determining the concentration of antagonist (IC2) required to reduce the effects of a 2 x dose of agonist to that of a single dose. The IC2 value was obtained from a plot of % response against dose of antagonist. No

assumptions were made concerning the kinetics of agonist/ antagonist interactions.

Materials [3H]-DAGOL (41 Ci mmol 1) and [3H]-DPDPE (31 Ci mmol -1) were from Amersham International p.l.c., [3H]-ethylketocyclazocine (EKC, 24 Ci mmol 1) and [3H]U69593 (60CimmolP ) were from New England Nuclear. [DAla2, MePhe4Gly(ol)5] enkephalin (DAGOL) and DPDPE were from Peninsula Labs, San Carlos CA; trans-(±)-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzacetamine methane sulphonate (U50488H) and (5,7,8)-±)-Nmethyl-N-(7-(1 -pyrrolidinyl- 1-oxaspiro(4,5)dec-8-yl)benzeneacetamide (U69593) were from Upjohn, Kalamazoo, Michigan; (±)-trans-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzo[b]-thiophene-4-acetamide hydrochloride (PD117302) was a gift from Dr J. Hunter, Park-Davis Research Unit, Cambridge; nor-binaltorphimine (nor-BNI) was from Research Biochemicals Inc; naloxone hydrochloride was from Sigma Chemical Company, Poole, Dorset; Hanks balanced salt solution was from Gibco Ltd., Paisley, Scotland.

Results Ligand binding experiments Parameters for the saturable binding of opioids to membranes from neonatal rat spinal cord are presented in Table 1. In these experiments p-binding sites were labelled with [3H]DAGOL, and K-sites with [3H]-U69593 or [3H]-EKC. Binding of [3H]-EKC to u-sites was blocked by including 100nM DAGOL in all samples. As before (Dray et al., 1989), we found approximately four times more y- than K-sites. There were also approximately twice as many K-like binding sites for [3H]-EKC than for [3H]-U69593. Saturable binding sites for the 6-ligand [3H]-DPDPE could not be detected. Slopes of Hill plots for [3H]-DAGOL and [3H]-U69593 binding were close to unity. The Hill plot for binding of [3H]EKC to nominally K-sites had a slope significantly less than one.

Results of competition experiments are shown in Table 2. Unlabelled DAGOL and naloxone competed for [3H]DAGOL sites much more effectively than the K-ligands U50488, U69593, PD1 17302 and nor-BNI, or the delta-ligand DPDPE (Table 2A). DPDPE did have a low, but measurable affinity for p-sites, consistent with a weak agonist activity at this receptor (Dray et al., 1989). Table 1 Parameters for binding of opioid ligands to membranes of neonatal rat spinal cord

Radioligand

[3H]-DAGOL [3H]-U69593 [3H]-EKC* [3H]-DPDPE

Kd (nM)

0.94 + 0.06 2.3 + 0.60 0.80 + 0.19 Not detectable

Bmax

(pmol mg1 protein)

slope

0.22 + 0.04 0.055 + 0.006 0.11 + 0.02

0.92 + 0.03 0.92 ± 0.03 0.72 ± 0.07

Hill

Saturable binding was measured as described in Methods. Parameters were estimated by non-linear regression on the binding isotherm, assuming a single type of non-interacting binding sites. Hill slopes are from linear regression on Hill plots. * Binding of [3H]-ethylketocyclazocine ([3H]-EKC) was measured in the presence of 100nM unlabelled [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAGOL), to prevent interaction at u-sites. Data are means ± s.e.mean from four to six experiments. [3H]-DPDPE = [3H]-[D-Pen2, D-Pen5] enkephalin.

OPIOIDS ON NEONATAL RAT SPINAL CORD Table 2 Competition for opioid binding sites in neonatal rat spinal cord membranes Competer

[3H]-DAGOL

Radioligand [3H]-U69593

[3H]-EKC*

1.0 + 0.21 > 1000 > 1000 900 + 52 78 + 7.0 2.1 + 0.07 620 + 14

400 + 12 0.86 + 0.06 0.82 + 0.46 0.49 + 0.04 0.78 + 0.10 3.5 + 0.10 > 1000

> 1000 18 + 4.4 24 + 10 31 + 10 5.8 + 2.2 120 + 46 > 1000

0.92 + 0.03

0.88 + 0.11 1.0±0.04 0.87 + 0.08 0.98 + 0.10 0.98 + 0.15 0.96 + 0.17

0.86 + 0.08 0.63 + 0.07 0.79 + 0.04 0.49 + 0.10 1.0+0.09

ICjO values (nM) DAGOL U50488 U69593 PD1 17302 Nor-BNI Naloxone DPDPE B Hill slopes DAGOL U50488 U69593 PD1 17302 Nor-BNI Naloxone DPDPE

A

1.0 + 0.03 0.88 + 0.01 1.2 + 0.10

IC50 values (the concentration of competing ligand required

to reduce saturable binding of the radioligand by 50%) and Hill slopes were estimated by linear regression on Hill plots of the competition data, and are presented as means + s.e.means from 3 to 6 experiments. * Measurements with [3H]-EKC were made in the presence of 100nm DAGOL to prevent interaction at p-sites. In the competition assays, saturable binding was at least 70% of total binding. Nor-BNI = nor-binaltorphimine; for key to other abbreviations see Table 1.

The K-ligands were much more potent than either DAGOL or DPDPE in competition for [3H]-U69593 sites. There were no large differences in affinity for these sites between Kligands. Notably U50488 was similar to U69593 and PD1 17302 in this assay. The antagonist nor-BNI had about 100 fold higher affinity for K- than for p-sites, confirming previous assessments of its selectivity (Takemori et al., 1988).

Once binding to p-receptors had been blocked with a high concentration of unlabelled DAGOL, neither DAGOL nor DPDPE competed for the remaining [3H]-EKC binding sites. Affinities of the K-ligands for these sites were generally lower than affinities for [3H]-U69593 binding sites. Again there was no difference between U50488 and the other K-ligands. Naloxone showed considerably lower affinity for the [3H]-EKC sites than for the [3H]-U69593 sites. Hill plots for competition against either [3H]-DAGOL or [3H]-U69593 all had slopes close to one. This was not found for sites labelled with [3H]-EKC. Notably nor-BNI and U69593 had Hill slopes considerably less than one in competition for these sites.

Spinal-cord/tail experiments Reproducible ventral root responses were obtained following stimulation of peripheral fibres with innocuous (brush) or noxious (heat or capsaicin) stimuli (Figure 1). Superfusion of the spinal cord with opioid agonists consistently produced a dose-related depression of capsaicin and heat evoked responses (Figures 1-3). The EC50 values are shown in Table 3. Overall the rank order of potency was DAGOL > U69593 = PD117302 > morphine > U50488 = DPLPE. Each opioid was more potent against the capsaicin-evoked response than against responses evoked by heat (Table 3). In some experiments (2 for U50488; 2 for U69593; 3 for P117302) where doses of K-agonists < 1nm were tested an initial enhancement of nociceptive responses was observed (Figure 3a). This phenomenon was not quantified or studied in detail, but similar observations have been made in vivo, following intrathecal administration of Kc-ligands (Knox & Dickenson, 1987). Subsequent higher agonist doses always produced a depression of the nociceptive response. The antagonist naloxone, was equally potent against the same opioid when compared in tests in which either capsaicin or heat was used to evoke the nociceptive response. The naloxone IC2 concentration was lowest (most potent) against the p-agonists DAGOL (Figure lb) and morphine, and the 5-

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Figure 1 The dose related depression of nociceptive spinal responses by [D-Ala2, MePhe4, Gly(ol)'] enkephalin (DAGOL) and the antagonism of these effects by naloxone. (a) The reproducible responses to capsaicin (Caps) and innocuous brush. DAGOL at 5 and 10nm selectively attenuated the nociceptive responses to capsaicin in a naloxone reversible manner. (b) Top traces show the reproducible spinal ventral root depolarization produced by stimulation of peripheral nociceptive fibres with heat (48°C) or capsaicin (0.5 pM). The middle traces show the attenuation of the nociceptive responses by cumulative spinal superfusion with DAGOL. Note that the capsaicin-evoked response appears to be attenuated preferentially. In the bottom traces superfusion with 2 nm naloxone reduced the depressant effect of 8 nM DAGOL to that produced by 4 nM DAGOL. This was the approximate IC2 concentration of naloxone. Drugs administered (indicated by the bar below the trace) in the spinal superfusate were allowed to equilibrate for at least 15 min before stimuli were repeated. The calibration bars are as indicated.

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I.F. JAMES et al. Table 3 Potency comp3arisons of opioids agonists against spinal responses evoked by peripheral stimulation with capsaicin or heat (480C)

Naloxone

IC2 (nM)

ED50 (nM) A Against capsaicin Morphine DAGOL DPLPE U50488 U69593 PD1 17302

56 2.8 120 120 5.5 4.8

10 5.8 7.6 >500 50 50.2

150 7.8 200 270 25 24

11 7.0 8.3 500 53 64

nor-BNI

IC2 (nM) > 500

50 63 60

B Against heat

Morphine DAGOL DPLPE U50488 U69593 PD117302

Freshly prepared solutions of nor-BNI consistently antagonised the effects of U50488 (8 of 10 experiments), U69593 (7 of 7 experiments, Figure 3b) and PD117302 (8 of 8 experiments) and similar IC2 concentrations were found whether heat or capsaicin was used as the stimulus (Table 3). The effect of DAGOL (4 of 4 experiments) was not reduced by nor-BNI superfused on the spinal cord up to a concentration of 500 nM.

>500 37 50 42

ED50 values were obtained from plotting % depression of the nociceptive response against drug concentration. Three to 5 concentrations were used for each estimate in each of 6-10 experiments. The IC2 values for naloxone and nor-BNI express the concentration of antagonist required to reduce the effect of a 2 x dose of opioid agonist to that of a single dose (n = 4-6 for each determination). For each determination at least three doses of antagonist were used, and the IC2 value was interpolated from a plot of % reduction in agonist effect against antagonist dose. For key to abbreviations used see Tables 1 and 2.

agonist DPLPE (Figure 2a). Naloxone also consistently antagonised the effect of U69593 and PD117302 (Figure 3a) though approximately ten fold higher concentrations were required (Table 3). In most experiments the naloxone IC2 concentration against U50488 was greater than 500 nm, arbitrarily chosen as the upper concentration limit for routine testing (Figure 2b, Table 3).

Discussion We have shown that there are functional g- and K-opioid receptors in neonatal rat spinal cord, and that activation of either of these receptors depresses the ventral root response evoked by noxious peripheral stimuli. p-Receptors were identified with the selective agonist DAGOL (Handa et al., 1981) and by showing that the effects of DAGOL were readily reversed by naloxone. K-Receptors were identified with the selective agonists U69593 (Lahti et al., 1985) and PD117302 (Clark et al., 1988). The actions of the K compounds were blocked by naloxone, but about ten times higher concentrations were needed than for blockade at p-receptors. The ten fold difference in sensitivity to naloxone betweeen M- and Kreceptors is well established in other tissues, for example the guinea-pig ileum and the mouse vas deferens (Lord et al., 1977; Goldstein & James, 1984). The actions of U69593 and PD117302 were also blocked by the K-selective antagonist nor-BNI, which was ineffective against DAGOL. These findings are in keeping with both in vitro (Birch et al., 1987; Portoghese et al., 1987; Takemori et al., 1988) and in vivo data (Sandler et al., 1988; Takemori et al., 1988; Tortella et al., 1989) showing nor-BNI to be a highly selective K-ligand. However, other studies suggest considerable difficulties in using this compound in vivo, as it exhibits effects only after a considerable delay and shows poor selectivity (Birch et al., 1987). A third well documented K-agonist, U50488 (Von Voigtlander et al., 1983), gave anomalous results. It was less potent than expected and very high concentrations of naloxone were

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Figure 2 Effects of [D-Pen2, L-Pen5]enkephalin (DPLPE) and U50488 on the spinal nociceptive responses. (a) Illustrates the doserelated depression of spinal responses by the 5-receptor ligand DPLPE (middle trace). The bottom traces show examples of the dose-related antagonism of DPLPE (300 nM) by cumulative doses of naloxone. (b) Shows the non-selective depression of nociceptive responses and innocuous brush responses produced by 300nM U50488. The effect of U50488 was not influenced by superfusion with 600 nM naloxone.

OPIOIDS ON NEONATAL RAT SPINAL CORD

507

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Heat Caps U69, 593 8 nM: nor-BNI 50 nM

Figure 3 Dose-related depression of the noiciceptive spinal response by K-agonists and the effects of antagonists. (a) The top traces show control responses to heat (480C) and capsaicin (Caps, 0.5 pM) administered to the tail. The middle traces show that superfusion of the spinal cord with PD1 17302 (indicated by the bar below the traces), for at least 15 min before peripheral stimuli were retested, produced a dose-related depression of the nociceptive response. The response evoked by capsaicin was more substantially depressed than that evoked by heat, and completely abolished by 16 nM PD117302. Note that the lowest dose of PD117302 (1 nM) produced an enhancement of the nociceptive responses. The bottom traces show antagonism of the PD1 17302 effect by concomitant superfusion of the spinal cord with naloxone (50nM). At this concentration the effect of the 16nM PD117302 was reduced to approximately that of 8 nm. The calibration bars are as indicated. (b) The top trace shows control responses to capsaicin and heat. Superfusion of the spinal cord with cumulative doses of U69593 produced a dose-related depression of the nociceptive responses (middle traces). A concomitant spinal superfusion of nor-binaltorphimine (nor-BNI, 50 nM) antagonized the effect of U69593, reducing the effect of 8 nm U69593 to that normally seen with 4 nM.

required to reverse its effects. The K-antagonist nor-BNI was effective against U50488 at the same concentrations as for other K-agonists. These results suggest that U50488 may be acting on a different population of receptors from U69593 and PD1 17302. DPLPE, which is normally a 6-selective agonist (Mosberg et al., 1983), also depressed the ventral root response, again at comparatively high concentrations. The actions of DPLPE were as sensitive to naloxone as those of DAGOL and morphine. In other systems, 6-agonists are about ten times less sensitive to naloxone than p-agonists (Lord et al., 1977; Goldstein & James, 1984). The low potency and relatively high sensitivity to naloxone are consistent with DPLPE acting at p-receptors in neonatal rat spinal cord, where binding assays suggest that there are no 6-sites (see below). Our binding data are consistent with single types of binding sites for [3H]-DAGOL and [3H]-U69593, in that Hill slopes for binding isotherms and competition assays were close to one. Hill slopes for [3H]-EKC binding and for competition assays with [3H]-EKC were less than one. There were more sites for [3H]-EKC than for [3H]-U69593, and the affinities of K-ligands for sites labelled with [3H]-EKC were lower than those for [3H]-U69593 sites. These results suggest that [3H]EKC labels more than one type of binding site and that at least some of the [3H]-EKC sites have a low affinity for U69593. Zukin et al. (1988) have described subtypes of Kreceptors in rat and guinea-pig brain based on heterogeneity of [3H]-EKC binding. In their model, Kl-sites bind PD117302, U69593 and U50488 with reasonable affinities. K2-sites have lower affinities than K,-sites for U50488 and PD117302, and do not bind U69593 even at high concentrations (up to 10pM). Sites for [3H]-U69593 in neonatal rat spinal cord could correspond to the K,-receptors proposed by Zukin and colleagues (1988). We could not identify the addi-

tional EKC sites as Zukin's c2-receptors because although the competition assays showed that U69593 had a relatively low affinity for these sites, so too did U50488 and PD117302. We have no evidence that heterogeneity of [3H]-EKC binding sites represents heterogeneity of c-receptors. The pitfalls of using non-selective radioligands in conjunction with unlabelled blocking ligands in these kind of experiments have been emphasized recently by Traynor (1989). We conclude from the binding and functional assays that pand K-receptors are present and are involved in the modulation of nociception in neonatal rat spinal cord. In fact, spinal antinociceptive actions of K-ligands are most easily demonstrated in neonatal and young animals (Allerton et al., 1989; Dickenson & Sullivan, personal communication), while in the adult, spinal mechanisms are relatively insensitive to K-opioids (Knox & Dickenson, 1987). The reasons for this are unclear. It is possible that c-receptors are involved in spinal developmental processes, and may be functionally less significant in adults. The binding assays provided no evidence for 6-sites. As discussed above, in the functional assays the naloxone sensitivity of DPLPE effects suggests that this normally 6-selective ligand can act at p-sites in tissues that lack 6-receptors. U69593 and PD117302 are c-agonists with high potency and efficacy. Although U50488 bound with high affinity to K-receptors, its potency as an antinociceptive agent was surprisingly low. We suggest that U50488 may have a relatively low efficacy at K-receptors. The difference in IC2 values for naloxone between U50488 and the other ic-agonists could then be explained if at the high concentration of U50488 required (because of the low efficacy) to activate c-receptors, other receptors were also activated. These would have to be relatively insensitive to naloxone and unaffected by either U69593 or PD117302 at the concentrations that we used. Others have also described effects of U50488 that are not sen-

w08

I.F. JAMES et al.

sitive to naloxone or the opioid antagonist 16methylcyprenorphine, and have concluded that U50488 can act at non-opioid receptors (Hayes et al., 1988; Leff & Dougal, 1989). Our data suggest that there may be a population of naloxone-insensitive receptors for U50488 in neonatal rat

cord, but that these sites are still sensitive to nor-BNI. If nor-BNI is highly selective for K-opioid receptors (and does not interact with non-opioid receptors) then these additional sites for U50488 would represent a subtype of K-receptor.

References ALLERTON, C.A., BODEN, P.R., HILL, R.G., HUGHES, J., HUNTER, J.C.

MOSBERG, H.I., HURST, R., HRUBY, V.J., GEE, K., YAMAMURA, H.I.,

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(Received May 29, 1989 Revised November 6, 1989 Accepted November 29, 1989)

Opioid receptor ligands in the neonatal rat spinal cord: binding and in vitro depression of the nociceptive responses.

1. Opioid receptors in the neonatal rat spinal cord have been characterized by measurements of ligand binding to crude membrane fractions and by funct...
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